SUMMARY RIG-I detects invading viral RNA and activates the transcription factors NF-κB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5′-triphosphate strongly activates the RIG-I–IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria and cytosol. Activation of RIG-I requires not only RNA, but also polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. RIG-I binds specifically to K63 polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63 ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a new ubiquitin sensor, and demonstrate that unanchored K63 polyubiquitin chains are signaling molecules in antiviral innate immunity.

TRAF6 is a ubiquitin ligase essential for the activation of NF-κB and MAP kinases in multiple signaling pathways including those emanating from the interleukin-1 and Toll-like receptors (IL-1R/TLR)1-3. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of Ubc13 and Uev1A to catalyze Lys-63 (K63)-linked polyubiquitination, which activates the TAK1 kinase complex4,5. TAK1 in turn phosphorylates and activates IκB kinase (IKK), leading to activation of NF-κB. Although several proteins are known to be polyubiquitinated in the IL-1R/TLR pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. Herein, we reconstituted TAK1 activation in vitro using purified proteins and found that free K63 polyubiquitin chains, which are not conjugated to any target protein, directly activated TAK1 through binding to the ubiquitin receptor TAB2. This binding leads to autophosphorylation and activation of TAK1. We also found that unanchored polyubiquitin chains synthesized by TRAF6 and Ubc5 activated the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a novel mechanism of protein kinase regulation.

Nuclear factor kappa enhancer binding protein (NF-kappaB) regulates diverse biological processes including immunity, inflammation, and apoptosis. A vast array of cellular stimuli converges on NF-kappaB, and ubiquitination plays an essential role in the coordination of these signals to regulate NF-kappaB activity. At least three steps in NF-kappaB activation directly involve ubiquitination: proteasomal degradation of inhibitor of NF-kappaB (IkappaB), processing of NF-kappaB precursors, and activation of the transforming growth factor (TGF)-beta-activated kinase (TAK1) and IkappaB kinase (IKK) complexes. In this review, we discuss recent advances in the identification and characterization of ubiquitination and deubiquitination machinery that regulate NF-kappaB. Particular emphasis is given to proteasome-independent functions of ubiquitin, specifically its role in the activation of protein kinase complexes and in coordination of cell survival and apoptosis signals downstream of tumor necrosis factor alpha (TNFalpha).

A growing number of agents targeting ligand-induced Wnt/β-catenin signaling are being developed for cancer therapy. However, clinical development of these molecules is challenging because of the lack of a genetic strategy to identify human tumors dependent on ligand-induced Wnt/β-catenin signaling. Ubiquitin E3 ligase ring finger 43 (RNF43) has been suggested as a negative regulator of Wnt signaling, and mutations of RNF43 have been identified in various tumors, including cystic pancreatic tumors. However, loss of function study of RNF43 in cell culture has not been conducted, and the functional significance of RNF43 mutations in cancer is unknown. Here, we show that RNF43 inhibits Wnt/β-catenin signaling by reducing the membrane level of Frizzled in pancreatic cancer cells, serving as a negative feedback mechanism. Inhibition of endogenous Wnt/β-catenin signaling increased the cell surface level of Frizzled. A panel of 39 pancreatic cancer cell lines was tested for Wnt dependency using LGK974, a selective Porcupine inhibitor being examined in a phase 1 clinical trial. Strikingly, all LGK974-sensitive lines carried inactivating mutations of RNF43. Inhibition of Wnt secretion, depletion of β-catenin, or expression of wild-type RNF43 blocked proliferation of RNF43 mutant but not RNF43-wild-type pancreatic cancer cells. LGK974 inhibited proliferation and induced differentiation of RNF43-mutant pancreatic adenocarcinoma xenograft models. Our data suggest that mutational inactivation of RNF43 in pancreatic adenocarcinoma confers Wnt dependency, and the presence of RNF43 mutations could be used as a predictive biomarker for patient selection supporting the clinical development of Wnt inhibitors in subtypes of cancer.T he evolutionarily conserved Wnt/β-catenin signaling pathway plays critical roles in embryonic development and adult tissue homeostasis (1, 2). Wnt signaling regulates the turnover of the transcription cofactor β-catenin and controls key developmental gene expression programs (3). In the absence of Wnt pathway activation, cytosolic β-catenin is degraded by the β-catenin destruction complex, consisting of adeomatous polyposis coli (APC), AXIN1/2, and glycogen synthase kinase 3α/β (GSK3α/β). Wnt ligand activates its two receptors, Frizzled and LRP5/6, and inactivates the β-catenin destruction complex. Stabilized β-catenin enters the nucleus, binds to the TCF family of transcription factors, and activates transcription. Secretion of Wnt proteins requires Porcupine (PORCN), a membrane bound O-acyltransferase dedicated to Wnt posttranslational acylation (4, 5). Precise regulation of Wnt signaling is critical and various feedback control mechanisms exist to ensure proper signaling output.Aberrant activation of Wnt/β-catenin signaling has been implicated in tumorigenesis, and many downstream components of the Wnt pathway are mutated in cancers (6). Truncation mutations of APC are found in 80% of colorectal cancer. Stabilization mutations of CTNNB1 (β-catenin) and loss of function mutations of AXIN1/2 are also fo...

SUMMARY RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.